Fig 1.

Phosphorylation of ASK1 at Ser967 is dynamically regulated. (a to f) COS7 cells were transfected with HA-ASK1 or HA-ASK1 S967A. (a) Ser967 phosphorylation was monitored after serum starvation for the indicated times. (b) Serum starvation-induced apoptosis was measured by staining with annexin V and 7-AAD after 24 h. The data represent means plus the standard deviations (n = 5). (c) COS7 cells were serum starved for 24 h and treated with the indicated doses of IGF-1, EGF, PDGF, TGF-β, or vehicle (PBS plus 0.1% BSA) for 20 min. (d and e) After 24 h of serum starvation, the cells were treated with IGF-1 or vehicle as indicated. (f and g) COS7 cells were treated with H₂O₂ (1 mM) in the presence of IGF-1 (100 ng/ml) or vehicle (PBS plus 0.1% BSA). The level of pSer967 of overexpressed (f) or immunoprecipitated endogenous ASK1 (g) was measured. pS967 and total ASK1 protein were detected by Western blotting (a, c to g) and quantified with ImageJ (g).